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Abstract

Background

IKK-2 is an important regulator of the nuclear factor-κB (NF-κB) which has been implicated
in survival, proliferation and apoptosis resistance of lymphoma cells. In this study,
we investigated whether inhibition of IKK-2 impacts cell growth or cytotoxicity of
selected conventional chemotherapeutic agents in non-Hodgkin's lymphoma.

Two established model systems were used; Follicular (WSU-FSCCL) and Diffuse Large
Cell (WSU-DLCL2) Lymphoma, both of which constitutively express p-IκB. A novel, selective
small molecule inhibitor of IKK-2, ML120B (N-[6-chloro-7-methoxy-9H-β-carbolin-8-yl]-2-methylnicotinamide) was used to perturb NF-κB in lymphoma cells.
The growth inhibitory effect of ML120B (M) alone and in combination with cyclophosphamide
monohydrate (C), doxorubicin (H) or vincristine (V) was evaluated in vitro using short-term culture assay. We also determined efficacy of the combination in vivo using the SCID mouse xenografts.

Results

ML120B down-regulated p-IκBα protein expression in a concentration dependent manner,
caused growth inhibition, increased G0/G1 cells, but did not induce apoptosis. There
was no significant enhancement of cell kill in the M/C or M/H combination. However,
there was strong synergy in the M/V combination where the vincristine concentration
can be lowered by a hundred fold in the combination for comparable G2/M arrest and
apoptosis. ML120B prevented vincristine-induced nuclear translocation of p65 subunit
of NF-κB. In vivo, ML120B was effective by itself and enhanced CHOP anti-tumor activity significantly
(P = 0.001) in the WSU-DLCL2-SCID model but did not prevent CNS lymphoma in the WSU-FSCCL-SCID
model.

Conclusions

For the first time, this study demonstrates that perturbation of IKK-2 by ML120B leads
to synergistic enhancement of vincristine cytotoxicity in lymphoma. These results
suggest that disruption of the NF-κB pathway is a useful adjunct to cytotoxic chemotherapy
in lymphoma.

Background

NHL is the fifth most common type of cancer in the US representing 4.5% of cancer
cases. Since the early 1970's the incidence of NHL has doubled [1]. It is a group of heterogeneous diseases resulting from malignant transformation
of lymphocytes. Eighty-five percent of NHLs are B-cells that can be broadly classified
as aggressive (50%) and indolent (40%). Diffuse Large B-cell NHL (DLBCL) is the most
common subtype (30%) of all lymphomas and is the prototype of aggressive but curable
NHL. Follicular lymphoma (FL) is the second most common subtype, representing 22%
and is the most common indolent NHL [2,3].

To date, there is no consensus concerning the best treatment algorithm, but combination
chemotherapy has been the mainstay for treatment of NHL. Incorporation of the anti-CD20
monoclonal antibody, Rituximab, has led to improvements in overall survival [4,5]. More than half of patients with DLBCL can be cured with combination of Rituximab
(R) and cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Incorporating
Rituximab into conventional chemotherapy for follicular lymphoma has lead to higher
response rates and longer durations of remission compared with chemotherapy alone
[6]. The success of Rituximab suggests that additional targeted therapeutics might improve
the efficacy of cytotoxic regimens.

Constitutively active NF-κB in lymphoma is known to induce resistance to intrinsic
and extrinsic apoptosis pathways [7]. NF-κB is a transcription factor comprised of homo- and heterodimers, p50/p105 (NF-κB1),
p52/p100 (NF-κB2), c-Rel, RelB, and p65 (RelA) [8]. Inhibitors of kappa B (IκBα, IκBβ and IκBε) contain ankyrin-like repeats that mediate
sequestration of NF-κB in the cytosol [9]. The interaction between IκBα and NF-κB is regulated by IκB kinase (IKK-1 and IKK-2).
Phosphorylation of IκBα leads to its degradation and release of NF-κB. NF-κB is then
able to translocate to the nucleus where it controls a number of molecules involved
in vital cellular functions, such as proliferation, apoptosis, and resistance to chemotherapy
[10-16].

Clinically, aberrant NF-κB activation has been linked to poor outcome in lymphomas
[17,18]. Therefore, these and other studies prompted us to investigate potential therapeutic
effects of inhibiting components of the NF-κB activation pathway in our lymphoma models.

Small molecule inhibitors (SMI) are used to selectively target molecules involved
in survival pathways. ML120B (N-[6-chloro-7-methoxy-9H-β-carbolin-8-yl]-2-methylnicotinamide) is a potent and selective inhibitor of IKK-2,
acting through blockade of the ATP-binding site in the kinase. ML120B has been shown
to inhibit tumor necrosis factor-α (TNF-α)-induced nuclear translocation of p65 subunit
of NF-κB and block TNF-α-stimulated cytokine production in human fibroblast-like synovial
cell cultures isolated from patients with rheumatoid arthritis [19]. ML120B inhibits both baseline and TNF-α-induced NF-κB activation in multiple myeloma
cells. It was also shown to inhibit the growth of multiple myeloma cells in vitro and in vivo SCID mouse models [20].

In this report, we show that ML120B inhibits the phosphorylation of IκBα, hinders
the growth of lymphoma cell lines in a concentration- and time-dependent manner and
reduces progression out of G0/G1 phase of the cell cycle. More importantly, ML120B
has a synergistic interaction with vincristine, a common cytotoxic agent used in the
treatment of hematological malignancies. Our studies suggest that IKK-2 inhibition
has a therapeutic role in lymphoma when used alone or in combination with cytotoxic
agents.

Results

Inhibition of IKK-2 Leads to Growth Inhibition of Lymphoma Cell Lines

In order to determine whether perturbation of the NF-κB activation pathway might play
a survival role in our lymphoma models, we seeded lymphoma cells in cluster plates
and treated the cells with ML120B at 0 to 80 μM. ML120B inhibited the growth of our
cells in a concentration- and time-dependent manner. After 48 hours of incubation,
the IC50 values were 18.8 and 23.2 μM for WSU-FSCCL and WSU-DLCL2 cells; respectively (Figure 1A). Accumulation of cells in sub G0/G1 fraction was not seen, indicating absence of
apoptosis (data not shown). Instead, there was a concentration-dependent increase
in cells arrested at G0/G1 and a reciprocal decrease of cells in S-phase (Figure 1B). ML120B (40 μM) induced a statistically significant increase of cells in G0/G1:
14% and 31% in WSU-FSCCL and WSU-DLCL2 (P = 0.02 and 0.01), respectively.

Figure 1.ML120B inhibits cell growth, cell cycle progression and phosphorylation of IκBα in
Lymphoma cell lines. A. IC50 was calculated by trypan blue exclusion assay for each cell line after 48 h incubation.
Cell number (vertical axis) = Mean and SEM [standard error of mean]. B. DNA content
was analyzed by PI staining using flow cytometry after 48 h for each cell line. C.
Lymphoma cell lines were cultured for 60 minutes with ML120B at the indicated concentrations
and whole cell lysates were subjected to Western blot for detection of p-IκBα, IκBα
and actin (Arrows indicate total IκBα).

The WSU-FSCCL and WSU-DLCL2 cells exhibit constitutive NF-κB activation as shown by the baseline expression of
the phosphorylated form of IκBα (p-IκBα). ML120B inhibited phosphorylated IκBα in
a concentration-dependent manner within one hour incubation with these cells. At 20
μM, ML120B inhibited p-IκBα by 100% in WSU-FSCCL and 40% in WSU-DLCL2 compared to control (Figure 1C).

Since CHOP is currently the standard regimen for lymphoma therapy, we chose to investigate
the effects of IKK-2 inhibition in combination with the cytotoxic components of CHOP
in WSU-FSCCL, i.e. cyclophosphamide monohydrate (C), doxorubicin (H), and vincristine
(V). The drug concentrations used were adopted from experiments in the WSU-DLCL2 cells as previously determined in our lab [21]. When used together, the IC50 for WSU-DLCL2 were as follows: C = 5.84 pM; H = 1.5 pM; and V = 260 pM. However, when these drugs
used individually against WSU-FSCCL in this study, we obtained less than 50% growth
inhibitions at 48 hours: C = 38% (i.e. IC40); H = 24% (i.e. IC25); and V = 39% (i.e. IC40). WSU-FSCCL cells were pre-incubated with ML120B for 1 hour at IC50 (18.8 μM) prior to addition of cytotoxic agents. The M/C combination did not induce
growth inhibition greater than the individual agents alone (Figure 2A & Table insert D). Similar results were obtained with M/H combination (Figure 2B & Table insert D). However, the M/V combination induced significant growth inhibition
greater than either agent alone (Figure 2C & Table insert D). To determine if this interaction is synergistic, WSU-FSCCL cells
were incubated with both agents at varying concentrations and the fractional effect
of both agents alone and in combination was calculated (Figure 2F). The M/V combination at 20 μM: 260 pM yielded a CI value of 0.225 which correlates
with "strong synergism". Increasing concentration of the agents yielded "very strong
synergism". Notably, M/V combination at lower concentration (10/130) also yielded
"synergism" (Table insert E in Figure 2).

When used alone against the WSU-FSCCL cells, vincristine at 520 pM induced G2/M arrest
at 24 hours which was released at subsequent time points (Figure 3A). Vincristine at its IC40 (260 pM) did not induce significant G2/M arrest compared to control. Higher concentrations
of vincristine (50 nM to 0.2 μM) were previously shown to induce G2/M arrest in cancer
cells [22,23]. Vincristine, at a concentration of 50 nM in our WSU-FSCCL cells induced a sustained
G2/M arrest over the 72 hour incubation period (Figure 3A). In the combination studies, the general effects on cell cycle were similar but
the magnitude was different depending on the concentration of the 2 compounds. The
most dramatic effect was seen with the higher concentrations (M 40 μM: V 520 pM) which
showed a significant increase in G2/M at 24 hrs at the expense of S and G0/G1 (Figure
3B). With further incubation (48 and 72 hrs), there was relative increase in G0/G1 suggesting
that cells arrested in G2/M at 24 hrs underwent apoptosis. In support of this interpretation
is the increase of cells in sub-G0 shown in Figure 3C. The combination of 40:520 M(μM): V(pM) induced a G2/M arrest at 24 hours that was
not statistically different from the 50 nM vincristine. The combination of the two
agents had a statistically significant concentration- and time-dependent increase
in apoptotic sub G0/G1fraction of cells. The increase in the apoptotic fraction induced
by the combination was higher compared to that found in both the control and 50 nM
vincristine at 24 hours (Figure 3C). At 48 hours, the M/V combination at 40 μM: 520 pM induced comparable apoptosis
to the higher concentration of single agent vincristine (50 nM). However, at 72 hours,
the high concentration of vincristine alone was more effective. Of special interest
is our observation that neither ML120B nor vincristine alone (at concentrations up
to 40 μM and 520 pM, respectively) induced sub G0/G1 accumulation (Figure 3D). Therefore, our data suggest that the M/V combination induces an initial G2/M arrest
at 24 hours, followed by apoptosis at 48 and 72 hours, leaving a fraction of unaffected
cells arrested in G0/G1. We used TUNEL assay to confirm that apoptosis occurred mostly
in G2/M. Figure 4A shows the increasing FITC positive population in treated cells compared with control
(horizontal axis). Total FITC positive cells also increased with increasing time of
incubation and concentration of M:V. For example, the total number of FITC positive
cells in the highest concentration of M:V combination (bottom line, panel B, Figure
4) increased from 12.75% at 24 h to 43.46% at 48 h and to 51.33% at 72 h. Moreover,
there was progressive shift-to-the-right of FITC positive cell population with increasing
incubation indicating increasing intensity of DNA breaks (apoptosis). Most of the
FITC positive cells were in G2/M phase followed by sub G0/G1 (which indicates late
apoptosis). Some apoptosis did occur from G0/G1 at 72 h. Induction of apoptosis was
also confirmed independently using 7-amino-actinomycin D (7-AAD) staining as shown
in Figure 5.

Figure 3.ML120B:vincristine combination induces cell cycle arrest and apoptosis. Flow cytometric analysis of cell cycle was done following PI staining of WSU-FSCCL
cells. A. WSU-FSCCL cells were exposed to vincristine (V) for the indicated times
and concentrations. B. WSU-FSCCL cells were pretreated with ML120B (M) for 60 minutes
followed by vincristine (V) at the indicated concentrations and times. C. Apoptotic
cells (Sub G0) analyzed by PI staining using flow cytometry in WSU-FSCCL for combinational
treatments, Mean and standard error of mean (SEM). D. Apoptotic cells analyzed as
in (C) in WSU-FSCCL cells treated with single agents.

Figure 4.ML120B:vincristine combination induces apoptosis in G2/M phase cells. WSU-FSCCL cells were pretreated with ML120B (M) for 60 minutes followed by vincristine
(V) at indicated concentrations in Panel B then analyzed for Terminal transferase
dUTP Nick End Labeling (TUNEL) staining using flow cytometry. A. Representative histograms
using the higher concentrations of M and V; upper panel: untreated, control cells;
lower panel: treated with ML120B (40 μM) and vincristine (520 pM). B. Full data of
FITC positive cells by cell cycle phase (sub G0, G0/G1, S, G2/M) according to PI staining
of DNA. The largest FITC positive population in treated cells was in G2/M followed
by sub G0.

Figure 5.7-AAD flow cytometric analysis of apoptosis. Representative scattergrams generated from 7-AAd staining of WSU-FSCCL treated with
vincristine at 520 pm (VCR), ML120B at 40 uM (ML) or the combination (ML + VCR) for
72 h (bottom panel left to right). Top panel left (+ control) are heat-treated dead
cells; top left (untreated) are live WSU-FSCCL cells from control culture. Within
each scattergram, 7-AAD (vertical axis) separates cells into dead (A, top), apoptotic
(B, middle) or live (C, bottom).

Mechanism of Interaction Between IKK-2 Inhibition and Vincristine

To define some of the molecular mechanisms by which ML120B synergizes with vincristine
in WSU-FSCCL cells, we first evaluated selected markers of apoptosis. There was only
minimal activation of apoptosis executioner (caspase-3) by each agent alone (Figure
6). However, the ML120B: vincristine combination induced caspase-3 cleavage. The combination
also significantly induced PARP cleavage. Both agents, individually and in combination,
enhanced the expression of cleaved caspase 8. These findings support the flow cytometry
data presented in Figures 3, 4, 5 showing that ML120B: vincristine combination induces significant apoptosis whereas
neither agent alone has significant effect at the concentrations used in this study.
These data also support the synergistic growth inhibitory effect shown in Figure 2. To explain such synergy, we evaluated the effects of different treatments on p65
in WSU-FSCCL cells by western blots and immunofluorescence. As shown in Figure 6, exposure of cells to ML120B led to retention of p65 in the cytosol and reduction
in nuclear p65. This finding is consistent with published data [19] and with our finding that ML120B inhibits the phosphorylation, and subsequent degradation,
of Iκ-B (Figure 1). Vincristine, on the other hand, decreased the cytosolic p65 expression indicating
p65 translocation to the nucleus and activation of NF-κB pathway. This finding is
consistent with published data demonstrating that vincristine and other microtubule
inhibitors activate NF-κB [24]. In the combination treatment where cells are exposed to ML120B for one hour prior
to vincristine, p65 was sequestered in the cytosol comparable to levels of ML120B-alone
treated cells. These findings were confirmed by immunofluorescence studies (Figure
7) and led us to hypothesize that ML120B synergizes with vincristine by preventing
vincristine-induced activation of NF-κB pathway. As a transcription factor, NF-κB
controls many molecules that are associated with resistance to programmed cell [3,17,18].

Figure 6.IKKβ inhibition in combination with Vincristine reduces p65 nuclear translocation
and induces apoptosis. WSU-FSCCL cells were incubated with ML120B or Vincristine alone, or pretreated with
ML120B (60 minutes) then cultured with Vincristine at indicated concentrations for
24 h. Total cell lysates were subjected to Western blot for detection of indicated
protein using actin as a loading control. Nuclear and cytosolic p65 protein fractions
were extracted from total cell lysates. GAPDH is used as a cytosolic loading control.

Figure 7.Immunofluorescence microscopy images of WSU-FSCCL cells. Top panel represents control cells after 48 h of culture; top left, tubulin staining;
top center, NF-κB p65; top right, overlay of tubulin and p65. Bottom panel represents images of WSU-FSCCL cells after 48 h of treatment with ML120B (40
μM) and vincristine (520 pM). ML120B was added 60 minutes prior to vincristine: Bottom
left are vincristine-alone treated cells showing disruption of microtubules and translocation
of p65 to the nucleus as indicated by yellow-orange color. Bottom center: ML120B plus
vincristine treatment showing sequestration of p65 in the cytoplasm in most cells.
Bottom right: is same as bottom center except with DAPI counterstain to demarcate
the nuclei and confirm absence of p65 from nuclei in most cells.

Antitumor Activity of ML120B in Lymphoma-bearing SCID Mice

Finally, we determined the efficacy of ML120B in our lymphoma-bearing xenograft SCID
mouse models. ML120B did not prevent WSU-FSCCL from infiltrating into the CNS in this
systemic model (data not shown). It was not possible, therefore to determine its systemic
efficacy since the usual cause of animal death is CNS lymphoma [25]. Conversely, ML120B delayed the growth of WSU-DLCL2 SC tumors. In Figure 8A, single day doses did not induce significant tumor growth delay. However, a 28-day
course showed significant delay in tumor growth compared to single day doses (P =
0.03) and to control (P = 0.04). To determine whether our in vitro combination findings correlated in vivo, we compared ML120B with CHOP at its MTD. Figure 8B, shows that CHOP and ML120B significantly reduced tumor load when given alone compared
to control (P = 0.003 and 0.006, respectively). ML120B: CHOP combination significantly
delayed tumor growth compared to control (P = 0.003), CHOP alone (P = 0.003), and
ML120B alone (0.001). This data indicate that IKK-2 inhibition potentiates conventional
cytotoxic chemotherapy effect in vivo.

Figure 8.In vivo activity of ML120B alone, and with CHOP in WSU-DLCL2-SCID xenograft mouse model, tumor
weights represent Mean and standard error of mean (SEM). A. Activity of ML120B as single agent: WSU-DLCL2 tumors were xenografted s.c. into 10 ICR SCID mice/group on day 0 and dosing was initiated
on day 7. ML120B was administered p.o. 120 mg/kg one dose on day 7 (× 1), 60 mg/kg
twice (BID) on day 7 (× 2), and 60 mg/kg BID for 28 days starting on day 7 (× 28).
Each point represents mean tumor weight of animal in each group ± SEM. B. ML120B plus
CHOP treatment: Xenografts were developed as in A and treatment started on day 7.
CHOP was administered at maximum tolerated doses (MTD) as previously defined (see
Material and Methods), ML120B was administered p.o. 60 mg/kg BID for 28 days (as in
A), ML120B in combination with CHOP at MTD.

Discussion

In this study we show that inhibition of IKK-2 by a small molecule inhibitor, ML120B,
enhances the cytotoxic effect of the microtubule inhibitor, vincristine in lymphoma
cells. IKK-2 inhibition leads to sequestration of p65 in the cytosol and prevention
of vincristine-induced nuclear translocation. It was previously shown that NF-κB activation
is involved in vincristine resistance [26]. This is believed to be due, at least in part, to the positive effect of NF-κB on
cell cycle progression [27].

There are multiple approaches to target IKK-2/NF-κB pathway. Several specific IKK-2
inhibitors are under development (reviewed by Karin et. al [28]). These inhibitors have a wide range of IC50 in relationship to inhibiting IKK-2. For example, the IC50 of SPC-839, PS-1145, and BMS-345541 are 62 nM, 0.15 μM and 0.3 μM, respectively. ML120B
inhibits IKK-2 at an IC50 of 62 nM. ML120B, in the nM range is highly specific to IKK-2, but is capable of inhibiting
IKKε and other enzymes at an IC50 greater that 100 μM [29]. Other novel SMIs, such as GS143 suppress IκBα ubiquitination, but not IκBα phosphorylation.
Thus, inhibition of NF-κB activation is as complex as the activation pathway itself
with multiple sites as targets for inhibition [30].

The present study makes several key observations regarding IKK-2 as a potential therapeutic
target in lymphoma. First, we demonstrated that inhibition of IKK-2 by ML120B can
cause growth inhibition in a concentration- and time-dependent manner. The cause of
the growth inhibition was due to the increase of cells in G0/G1 phase of the cell
cycle. Our results suggest that ML120B alone acts by blocking cell growth and not
via apoptosis. Second, we demonstrated that ML120B can inhibit constitutive activation
of NF-κB in indolent and aggressive lymphoma cell lines in a concentration dependent
manner similar to what was observed in myeloma cells [20]. These observations suggest a broad application of IKK-2 inhibition in lymphoid tumors.

Interestingly, our data shows that IKK-2 inhibition synergizes the cytotoxic effects
of microtubule inhibitor, vincristine. This synergy was found at 1/100 the dose of
vincristine alone required to induce comparable G2/M arrest and apoptosis (520 pM
in combination with ML120B (40 μM) vs 50 nM when used alone, Figure 3A-C). Furthermore, our results suggest that the ML120B: vincristine combination induces
cell cycle arrest followed by apoptosis out of G2/M. Vincristine is a microtubule
depolymerizing agent. It was shown that depolymerization of microtubules activates
NF-kB and induces NF-kB-dependent gene expression [31]. Our data indicate that prevention of vincristine-induced nuclear translocation of
p65 and activation of NF-κB is a major mechanism of synergy between IKK-2 inhibition
and vincristine. This synergy is selective since we did not observe similar interaction
between IKK-2 inhibition and cyclophosphamide or doxorubicin. Cell death induced by
the ML120B: vincristine combination is through the apoptosis pathway since there was
evidence for caspase 3 and PARP cleavage (Figure 6). Constitutive activation of NF-κB in lymphoma and consequent activation of downstream
molecules like cIAP2 [32], p21 [33], and Bcl-2 [12] increases the threshold for apoptosis. This cell survival mechanism is accentuated
by exposure of cells to vincristine [24,33]. IKK-2 inhibition, by sequestering NF-κB in the cytosol and consequent down regulation
of pro-survival molecules, lowers the threshold of apoptosis in response to cytotoxic
agents like vincristine (Figures 3, 4, 5).

In vivo, we showed that ML120B: CHOP combination was well tolerated by the animals and induced
higher anti-tumor efficacy compared with each agent alone in our WSU-DLCL2-SCID model (Figure 8). We have previously shown that genistein (30 μM) sensitizes DLCL2 cells to CHOP [24]. Bharti et al., have shown that curcumin, a natural inhibitor of NF-κB, may sensitize
the cytotoxic effects of vincristine (50 μM) [34]. Sanda et al., showed that IKK inhibition by ACHP (10 μM) led to growth inhibition
of MM cells and potentiation of vincristine cytotoxicity [35].

Conclusion

In summary, our study shows the feasibility of inhibiting a constitutively active
NF-κB pathway in lymphoma cells. Such inhibition is associated with therapeutically
beneficial biological effects in vitro and in vivo. When used alone, ML120B elicited modest therapeutic gains. However, there was significant
synergy with the microtubule inhibitor, vincristine. Our data indicate that approaches
to NF-κB pathway inhibition are best used in combination with cytotoxic chemotherapy
rather than single agents. The major future challenge is to develop a more effective
IKK-2 inhibitor with lower cellular IC50 in order to make them more attractive clinically.

Materials and methods

Cell Culture and Reagents

The cell lines used in the study have been previously described; Follicular Lymphoma
(WSU-FSCCL) [36] and Diffuse Large Cell Lymphoma (WSU-DLCL2) [37]; The WSU-FSCCL cell line has been karyotyped at least 4 times since our initial publication
in 1993. The recent analysis in September of 2009 revealed the same chromosomal abnormalities
as previously reported; 47,XY,+der(1)i(1)(q10)del(1)(q32),t(1;13)(p31;q12),del(6)(q21q27),t(8;11)(q24;q22),t(14;18)(q32;q21).
The WSU-DLCL2 has been similarly karyotyped several times since its establishment
in 1990. The cell line acquired an additional abnormality, add(8q24), that was detected
for the first time in 1997. Since then the karyotype profile has remained stable with
no further changes. The most recent karyotype in September of 2009 revealed: 48,XY,t(1;2)(p36;q37),der(3)t(3;7)(q13;p15),t(4;14)(q27;q32),+7,i(7)(p10),der(7)t(3;7)(q21;p11.2),+8,add(8)(q24),t(14;18)(q32;q21),del(15)(q26.1),del(16)(q22)[10]. Furthermore, fluorescent in situ hybridization (FISH) using LSI MYC dual color break-apart
DNA probe (Vysis Inc.) revealed a deletion of the telomeric 3' region of CMYC gene
most likely due to unbalanced translocation affecting the CMYC gene region. Cells
were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum
(FBS), 1% L-glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin and incubated
at 37°C in a humidified incubator with 95%/5% CO2. Primary antibody specific for Actin was obtained from Santa Cruz Biotechnology,
(Santa Cruz, CA). Primary antibodies specific for Caspase-3, Caspase-9, PARP, p-IκBα
and IκBα were obtained from Cell Signaling, (Danvers, MA). G3PDH was obtained from
Trevigen, Inc (Gaithersburg, MD). Protein concentrations were determined using the
Micro BCA protein assay (Pierce Chemical Company, Rockford, IL). Cyclophosphamide
monohydrate was obtained from Mead Johnson (Evansville, IN). Doxorubicin hydrochloride
was obtained from Bedford Inc (SA, Australia). Vincristine was obtained from Pharma
Inc. (Bloomington, MN). ML120B was synthesized by Millennium Pharmaceuticals, Inc
(Cambridge, MA) and dissolved in DMSO. Concentration of DMSO in the final culture
was 0.44%.

Flow Cytometric Analysis of Cell Cycle and Apoptosis

Cell cycle analysis and sub G0/G1 DNA content were determined by flow cytometry using
propidium iodide (PI) staining. Cells were grown in the presence or absence of ML120B
or vincristine then centrifuged and washed. The cells were then fixed with 75% ice-cold
ethanol overnight and stained with 50 μg of PI and analyzed. To determine DNA fragmentation
(as indication of apoptosis) induced by treatment agents, we utilized standard terminal
deoxynucleotidyl transferase of dUTP nick end labeling (TUNEL) assay and propidium
iodide (PI) staining. The kit used in this method (ApoDirect In Situ DNA Fragmentation
Assay Kit, BioVision, Mountain View, CA; Catalog #K402-50) utilizes terminal deoxynucleotidyl
transferase (TdT) to catalyze incorporation of DUTP at the 3'-hydroxyl ends of the
fragmented DNA. The fluorescein-labeled DNA was detected by flow cytometry (horizontal
axis in Figure 4). PI staining was simultaneously used to separate cells into G0/G1, S, G2 M and sub-G0
compartments based on DNA content (vertical axis, Figure 4). The dual staining (dUTP and PI) allowed us to assign dUTP-positive cells to a cell
cycle phase. In this method, it is accepted that dUTP-positive cells are considered
apoptotic [38]. To confirm induction of apoptosis, we stained WSU-FSCCL cells with 7-AAD as previously
published from our laboratory [39]. All flow cytometry analysis of cells was done on FACScan (Becton-Dickinson, San
Jose, CA).

Fluorescence Microscopy

WSU-FSCCL cells, treated and untreated, were harvested, washed once with PBS and fixed
for 10 min with 3.7% formaldehyde in PBS. All procedures were carried out at room
temperature. Following fixation, cells were washed 3 times with PBS, blocked for 45
min with 0.5% BSA in PBS and then incubated for 3 hr in 200 μl PBS containing 0.1%
saponin (PBS-S), 1 μg/ml each of two primary antibodies, mouse anti-human NF-κBp65
and rabbit anti-tubulin. After incubation with primary antibodies, cells were carefully
washed 3 times with PBS-S and then resuspended in PBS-S containing 5% goat sera and
10 μg/ml each of two fluorescently-labeled secondary antibodies and DAPI (10 μg/ml)
for nuclear staining, if used. Cells were incubated for 1 hour, washed X3 with PBS-S
and then fixed for 1 min with 3.7% formaldehyde. Following the final fixation, cells
were washed 3 times with PBS containing no saponin. Cell suspensions were mounted
on 1% gelatin-coated slide, dried, sealed with coverslips and visualized using an
Olympus BX40 microscope equipped with laser light and fluorescence filter cubes for
UV, green and red fluorescence. Visual recordings were captured separately using an
RT-Spot Color Camera (Diagnostic Instruments, Inc, Sterling Heights, MI) and merged
using Super Spot software (Diagnostic Instruments) to complete the overlay and final
pictures.

Establishment and Propagation of Xenografts

3-4 week old female ICR mice with severe combined immune deficiency (SCID) were purchased
from Taconic Farms (Germantown, NY). Animals were housed in special protective environment
and left to adapt for few days before beginning the experiments. To initiate the WSU-DLCL2-SCID xenografts, (5-10) × 106 WSU-DLCL2 cells in serum-free RPMI 1640 medium were injected subcutaneously (SC) in the flank
areas of each animal. Palpable tumors were detected by clinical examination in about
2 weeks. When tumor weight reached 1000-1500 mg, animals were euthanized; tumors dissected
out, placed in RPMI 1640 medium in sterile environment and minced into small fragments
(20-30 mg each). To propagate the xenografts, tumor fragments were implanted SC, using
a trocar, into flanks of 3-4 week old female ICR-SCID mice. Forty animals were implanted
with WSU-DLCL2 tumors for the single agent (ML120B alone) experiment and forty for the combination
study (CHOP plus ML120B). The WSU-FSCCL-SCID is a systemic model which is established
by injecting 107 WSU-FSCCL cells in serum-free medium intravenously via tail vein of ICR-SCID mice.
The growth pattern and assessment of response of this model to ML120B were the same
as previously published from our laboratory [25].

Efficacy Trial Design

WSU-DLCL2 tumor-bearing animals were randomly assigned to control or one of 3 treatment dose/schedules
of ML120B; 10 animals in each group. Therapy was started one week after tumor implantation.
Group 1 received one dose of ML120B at 120 mg/kg. Group 2 received 60 mg/kg twice
(every 12 hours). Group 3 received 60 mg/kg twice a day for 28 days. All treatments
were given through oral gavage. ML120B compound was dissolved in 5% (hydroxypropyl)
methylcellulose. Control group animals received vehicle alone. CHOP MTD in SCID mice
was previously determined in our laboratory [21] for one injection (i.e.40 mg/kg, i.v. cyclophosphamide; 3.3 mg/kg,i.v. doxorubicin;
0.5 mg/kg,i.v. vincristine; and 0.2 mg/kg orally prednisone every day for 5 days).
Animals were monitored 3 times per week for signs of toxicity, weight changes and
tumor measurements. They were euthanized to avoid discomfort if the tumor burden reached
~2000 mg (approximately 10% of the body weight). All animal experiments were done
according to protocols approved by the Animal Investigation Committee (AIC) of Wayne
State University.

Statistical Analysis

Statistical significance of drug-treated versus control measurements was determined
by the student t-test. The interaction between ML120B and vincristine was analyzed
using Calcusyn V2 software program to determine if the combinations were synergistic.
Calcusyn is based on the Chou-Talalay method [40], which calculates a combinational index (CI) to indicate synergistic effects where
CI < 0.9, is considered synergistic. Survival functions were estimated using the Kaplan-Meier
method and compared by the log-rank test. P-values <0.05 were considered statistically
significant.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AK had overall supervision of the project, data analysis and manuscript writing. AAA
did technical conduct of in vitro experiments and manuscript writing. AA did the animal
experiments. AS did p65 blots and assisted in immunofluorescence experiments and experimental
design. PS provided the ML120B IKK-2 inhibitor and data on binding to its target;
critiqued manuscript. AM re-characterized the WSU-FSCCL and WSU-DLCL2 cell lines used
in the study by cytogenetics and FISH; compared data with previous genetic profiles
of same cell lines. FB did the immunofluorescence experiments; participated in manuscript
writing and critique. RM supervised Dr. Arnold; Animal data analysis; review and critique
of the manuscript.